Idiopathic pulmonary fibrosis (IPF) is a progressive, chronic interstitial lung disease associated with high mortality (median survival of newly diagnosed patients is ˜3 years) and a uniformly poor prognosis (Khalil et al., “Idiopathic Pulmonary Fibrosis: Current Understanding of the Pathogenesis and the Status of Treatment,” CMAJ 171(2):153-60 (2004)). IPF is the pathological hallmark of interstitial lung diseases (Green, F. H., “Overview of Pulmonary Fibrosis,” Chest 122(6 Suppl):3345-3395 (2002)) and is characterized by increased deposition of extracellular matrix (ECM), including collagen. This lethal lung disorder presents a major clinical challenge, since effective therapeutic agents for reversing lung fibrosis have not yet been discovered (Phan et al., “The Myofibroblast as an Inflammatory Cell in Pulmonary Fibrosis,” Curr. Top. Pathol. 93:173-82 (1999)). The current hypothesis is that IPF represents a chronic injury/repair response to specific environmental insults (such as silica or asbestos). However, the precise molecular mechanisms underlying persistent fibroblast activation remains poorly understood. Myofibroblasts are now recognized as major effector cells in pulmonary fibrosis. They are characterized by the expression of α-smooth muscle actin (α-SMA), enhanced proliferation, and synthesis of ECM proteins (Sheppard et al., “Transcriptional Activation by NF-κB Requires Multiple Coactivators,” Mol. Cell. Biol. 19(9):6367-78 (1999)), and are thought to be derived from fibroblasts via the activity of TGF-β and other stimuli (Roy et al., “Regulation of α-Smooth Muscle Actin Gene Expression in Myofibroblast Differentiation from Rat Lung Fibroblasts,” Int'l J. Biochem. Cell Biol. 33(7):723-34 (2001)). In pulmonary fibrosis, myofibroblasts acquire resistance to apoptosis, which may account for the increased number of these cells present in fibroblastic foci (Horowitz et al., “Combinatorial Activation of FAK and AKT by Transforming Growth Factor-β1 Confers an Anoikis-Resistant Phenotype to Myofibroblasts,” Cell Sign. 19(4):761-71 (2007)).
Myofibroblasts are crucial effector cells in lung fibrosis, and derive from epithelial to mesenchymal transition (EMT) (Bedi et al., “Epithelial-to-Mesenchymal Transition and Chronic Allograft Tubulointerstitial Fibrosis,” Transplant. Rev. 22(1):1-5 (2008)), circulating fibrocytes (Andersson-Sjoland et al., “Fibrocytes Are a Potential Source of Lung Fibroblasts in Idiopathic Pulmonary Fibrosis,” Int. J. Biochem. Cell Biol. 40(10):2129-40 (2008)), and by self renewal in response to lung injury or chronic inflammation induced by stimuli such as silica and bleomycin. These myofibroblasts are accumulated in the injured lung and block alveolar gas exchange. Avoiding the formation of over-myofibroblasts would provide protection against lung fibrosis, and this could be achieved by inhibition of α-SMA expression in lung fibroblasts (Meyer-Ter-Vehn et al., “Lovastatin Inhibits TGF-β-Induced Myofibroblast Transdifferentiation in Human Tenon Fibroblasts,” Invest. Ophthalmol. Vis. Sci. 49(9):3955-60 (2008)).
After inhalation of pro-fibrotic stimuli (e.g. asbestos, silica, bleomycin) alveolar macrophages produce cytokines including TGF-β1 and TNF-α) that contribute to lung inflammation and fibrosis via different mechanisms (Hardie et al., “Conditional Expression of Transforming Growth Factor-α in Adult Mouse Lung Causes Pulmonary Fibrosis,” Am. J. Physiol. Lung Cell. Mol. Physiol. 286(4):L741-49 (2004); Liu et al., “Transforming Growth Factor-β1 Overexpression in Tumor Necrosis Factor-α Receptor Knockout Mice Induces Fibroproliferative Lung Disease,” Am. J. Respir. Cell Mol. Biol. 25(1):3-7 (2001)). TGF-β1 plays an essential role in wound healing and matrix molecule deposition. It induces myofibroblast differentiation and alveolar remodeling in vivo (Leask & Abraham, “TGF-β Signaling and the Fibrotic Response,” Faseb. J. 18(7):816-27 (2004); Lee et al., “Early Growth Response Gene 1-Mediated Apoptosis Is Essential for Transforming Growth Factor β1-Induced Pulmonary Fibrosis,” J. Exp. Med. 200(3):377-89 (2004)), and overexpression of this potent profibrotic mediator leads to progressive fibrosis in mice, with minimal inflammation (Sime et al., “Adenovector-Mediated Gene Transfer of Active Transforming Growth Factor-β1 Induces Prolonged Severe Fibrosis in Rat Lung,” J. Clin. Invest. 100(4):768-76 (1997)). TNF-α also contributes to lung fibrosis (Ortiz et al., “Expression of TNF and the Necessity of TNF Receptors in Bleomycin-Induced Lung Injury in Mice,” Exp. Lung Res. 24(6):721-43 (1998)), and its effects may be mediated through activation of other growth factors. TNF-α may also regulate the balance between cell survival and cell death. For example, TNF-α-deficient mice are protected against bleomycin-induced lung inflammation via reduced apoptosis of inflammatory cells (Kuroki et al., “Repression of Bleomycin-Induced Pneumopathy by TNF,” J. Immunol. 170(1):567-74 (2003)) Inhibition of TNF-α with infliximab may stabilize the progression of pulmonary fibrosis associated with collagen vascular disease (CVD) (Antoniou et al., “Infliximab Therapy in Pulmonary Fibrosis Associated with Collagen Vascular Disease,” Clin. Exp. Rheumatol. 25(1):23-28 (2007)).
Yin Yang 1 (YY1) is a ubiquitously expressed zinc finger transcription factor that can either activate or repress gene transcription, and plays an important role in cellular proliferation, differentiation, and apoptosis. Growing evidence indicates that YY1 contributes to the pathogenesis of cancer and inflammation (Austen et al., “Characterization of the Transcriptional Regulator YY1. The Bipartite Transactivation Domain Is Independent of Interaction with the TATA Box-Binding Protein, Transcription Factor IIB, TAFII55, or cAMP-Responsive Element-Binding Protein (CBP)-Binding Protein,” J. Biol. Chem. 272(3):1709-17 (1997); Gordon et al., “Transcription Factor YY1: Structure, Function, and Therapeutic Implications in Cancer Biology,” Oncogene 25(8):1125-42 (2006)). For example, YY1 negatively regulates p53, a tumor suppressor gene (Sui et al., “Yin Yang 1 Is a Negative Regulator of p53,” Cell 117(7):859-72 (2004)), and promotes tumor cell survival in part by preventing apoptosis (Huerta-Yepez et al., “Involvement of the TNF-α Autocrine-Paracrine Loop, via NF-κB and YY1, in the Regulation of Tumor Cell Resistance to Fas-Induced Apoptosis,” Clin. Immunol. 120(3):297-309 (2006); Vega et al., “Rituximab (Chimeric anti-CD20) Sensitizes B-NHL Cell Lines to Fas-Induced Apoptosis,” Oncogene 24(55):8114-27 (2005)). TNF-α-induced YY1 represses Fas expression, providing a mechanism whereby YY1 contributes to TNF-α-induced cell survival (Huerta-Yepez et al., “Involvement of the TNF-α Autocrine-Paracrine Loop, via NF-κB and YY1, in the Regulation of Tumor Cell Resistance to Fas-Induced Apoptosis,” Clin. Immunol. 120(3):297-309 (2006)). In fibroblasts, TNF-α induces YY1 in an NF-κB-dependent manner (Wang et al., “NF-κB Regulation of YY1 Inhibits Skeletal Myogenesis Through Transcriptional Silencing of Myofibrillar Genes,” Mol. Cell Biol. 27(12):4374-87 (2007)), supporting a link between the NF-κB pathway and YY1 expression (Huerta-Yepez et al., “Involvement of the TNF-α Autocrine-Paracrine Loop, via NF-κB and YY1, in the Regulation of Tumor Cell Resistance to Fas-Induced Apoptosis,” Clin. Immunol. 120(3):297-309 (2006); Vega et al., “Rituximab (Chimeric anti-CD20) Sensitizes B-NHL Cell Lines to Fas-Induced Apoptosis,” Oncogene 24(55):8114-27 (2005); Lei et al., “p38 MAPK-Dependent and YY1-Mediated Chemokine Receptors CCR5 and CXCR4 Up-Regulation in U937 Cell Line Infected by Mycobacterium tuberculosis or Antinobacillus actinomycetemcomitans,” Biochem. Biophys. Res. Commun. 329(2):610-15 (2005)). YY1 can bind to and activate type I and type II collagen gene promoters in fibroblasts (Riquet et al., “YY1 Is a Positive Regulator of Transcription of the Collal Gene,” J. Biol. Chem. 276(42):38665-72 (2001); Miao et al., “Identification of Two Repressor Elements in the Mouse α2(I) Collagen Promoter,” Arch. Biochem. Biophys. 361(1):7-16 (1999)), and also enhance fibronectin gene expression (Du et al., “Transcriptional Up-Regulation of the Delayed Early Gene HRS/SRp40 During Liver Regeneration. Interactions Among YY1, GA-Binding Proteins, and Mitogenic Signals,” J. Biol. Chem. 273(52):35208-15 (1998)). The expression of cyclooxygenase-2 (which also contributes to lung fibrosis) is also regulated by YY1 in macrophages (Joo et al., “Yin Yang 1 Enhances Cycloxygenase-2 Gene Expression in Macrophages,” Am. J. Physiol. Lung Cell. Mol. Physiol. 292(5):L1219-26 (2007)). These reports suggest that YY1 may play a role in fibrotic responses in the lung or other organs, but very little is known about the expression or function of YY1 in fibrotic conditions in vivo.
Experiments employing conditional deletion mouse stains revealed a crucial role for YY1 in the proliferation and differentiation of B lymphocytes (Liu et al., “Yin Yang 1 Is a Critical Regulator of B-Cell Development,” Genes Dev. 21(10):1179-89 (2007)) and oligodendrocytes (He et al., “The Transcription Factor Yin Yang 1 Is Essential for Oligodendrocyte Progenitor Differentiation,” Neuron 55(2):217-30 (2007)). Embryonic fibroblasts from YY1-deficient mice demonstrated reduced proliferation in vitro in proportion to the levels of YY1 protein expression. This finding indicates that YY1 controls fibroblast proliferation in a gene dosage-dependent manner (Affar el et al., “Essential Dosage-Dependent Functions of the Transcription Factor Yin Yang 1 in Late Embryonic Development and Cell Cycle Progression,” Mol. Cell Biol. 26(9):3565-81 (2006)). However, very little is known about the expression or function of YY1 in fibrotic conditions or disorders, such as lung fibrosis.
The present invention is directed to overcoming these and other deficiencies in the art.